Immunological methods for the treatment of gastrointestinal cancer

ABSTRACT

A method of treating gastrointestinal cancers dependent on the prohormones amidated gastrin- 17  and glycine extended G- 17 , comprising the administration to the patient of an anti-gastrin  17  immunogen which induces antibodies which bind and neutralize amidated and glycine-extended gastrin- 17.

BACKGROUND OF THE INVENION

The mature gastrin hormone occurs in two molecular forms which are namedwith respect to the number of amino acids in the peptide, i.e.,tetratriacontagastrin (G34) and heptadecagastrin (G17). In gastrinproducing cells, these gastrin hormones are posttranslationallyprocessed from a common precursor molecule termed “preprogastrin”, whichcontains a signal peptide. The signal peptide “pre” is removed in theendoplasmic reticulum of the cell, resulting in the “progastrin”peptide, which is in turn further processed in the cell to yield themature gastrins G34 and G17, before they are secreted into thebloodstream (Dickinson 1991). (The full citations for the referencescited herein are provided in the Reference Section preceding theclaims). The mature forms of G34 and G17 are both amidated (NH2) attheir carboxy terminal end. It has been elucidated that there aremultiple forms of G17 resulting from differential processing of theprecursor molecule, each of which may have different biologicalactivities (Dickinson 1995 and Ciccotosto et al. 1995).

The hormone gastrin is now a well recognized growth factor for humancolorectal adenocarcinomas (see Watson et al. 1993 for a review).Elevated plasma levels of total gastrin occurs in patients withcolorectal cancers, and in particular, increased amounts of the hormoneprecursor, progastrin, have been detected in many colorectal tumorsusing gastrin antisera (Ciccotosto et al. 1995).

Generally, in tumors such as those present in gastrin-dependent coloncancer, the cancer cells lose the ability to process prohormones tocompletion due to defects in the regulatory pathways of hormonesecretion. This leads to the production and secretion of differentmolecular forms of the hormone. Colon carcinoma cells do not efficientlyprocess progastrin and thus, produce mostly incomplete or aberrantgastrins, which results in less conversion of precursor gastrin to themature peptides (Dickinson 1993 and Rehfeld et al. 1993). The increasedgastrin level in colorectal tumors is, in part, attributed to theaberrant expression of the gastrin gene in the colorectal tumor cells(Hoosein et al. 1990, Baldwin et al. 1992 and Finley et al. 1993).Gastrin-like peptides have been identified in such cells (Hoosein et al.1988, Watson et al. 1991 and Finley et al. 1993), and were confirmed tobe precursor gastrin species (Van-Solinge et al. 1993 and Nemeth et al.1993).

Serum-associated G17 has the potential to stimulate the growth ofcolorectal tumors in an endocrine manner mediated by CCKB/gastrinreceptors (Watson et al. 1993). Gastrin-17 appears to be particularlyimplicated in stimulating the growth of human colorectal adenocarcinomasdue to a possible increased affinity for gastrin/cholecystokinin (CCK) Breceptors on the tumor cells, over other gastrin hormone species(Rehfeld, J. F. 1972). The CCKB/gastrin receptors were found to beexpressed in a high affinity form on 56.7% of human primary colorectaltumors (Upp et al. 1989). It has been postulated that a potentialautocrine loop may also exist due to endogenous production of precursorgastrin peptides by such tumors (Van-Solinge et al. 1993 and Nemeth etal. 1993), as it has recently been shown that the precursor gastrinmolecule, glycine-extended gastrin 17 (G17-Gly), stimulated the growthof a gastrointestinal tumor cell line. The trophic effects of G17-Gly ontumors has been shown to be mediated by a receptor other than theCCKB/gastrin receptor and an autocrine growth loop, possibly involvinggastrin precursors, has been postulated to be involved in theproliferation of gastrointestinal tumors (Seva et al. 1994).

Available treatments for tumors stimulated or induced by gastrin, andfor tumors that produce gastrin consists primarily of surgical resectionof the cancerous tissue. This approach is frequently unsuccessful; inmany instances, the tumors cannot be located or are present in anatomicsites that are inoperable. In most instances, these tumors do notrespond well to radiation or chemotherapy regimens, and new treatmentsare needed to supplement present procedures.

A number of high affinity CCKB/gastrin receptor antagonists have beendescribed, such as L-365,260 (Bock et al. 1989) and CI-988 (Hughes etal. 1990), which have been shown to effectively neutralize the effectsof exogenous gastrin on gastrointestinal tumor growth both in vitro andin vivo (Watson et al. and Romani et al. 1994). However, the antagonistslack specificity as they block the actions of all the potential ligandsof the receptor, such as gastrin-34 (G34) and CCK. Moreover, thecellular receptors which recognize and bind the gastrin precursor,G17-Gly, do not bind all the inhibitors tested (Seva et al. 1994). Thus,if a distinct receptor is involved in the autocrine growth cascade, thenthe gastrin antagonists may be unable to block this mechanism of tumorgrowth promotion.

A therapeutic method of selectively immunologically neutralizing thebiological activity of the gastrin hormone would provide an effectivemeans of controlling or preventing the pathologic changes resulting fromexcessive gastrin hormone production.

Co-assigned U.S. Pat. Nos. 5,023,077 and 5,468,494 disclose immunogeniccompositions useful for controlling G17 and G34 levels in a patient bygenerating anti-gastrin antibodies, and the use of such compositions forthe treatment of gastric and duodenal ulcers and gastrin-inducedcancers. The present invention concerns the use of the anti-Gl7immunogenic compositions disclosed in the Patent Nos. 5,023,077 and5,468,494 in the therapy of cancers whose growth is stimulated byprecursor glycine-extended and amidated gastrin 17.

The method of cancer therapy described in this invention has severaladvantages over present treatment methods. The method is non-invasive,selectively reversible, does not damage normal tissue, does not requirefrequent repeated treatments, and does not cross the blood brainbarrier.

SUMMARY OF THE INVENTION

The present invention provides immunological methods for the treatmentof gastrin-dependent tumors which comprise the active or passiveimmunization of a patient with anti-G17 immunogen or antibodies againstgastrin 17 hormone in order to control the patient's glycine-extendedand amidated gastrin 17 levels. By inducing anti-gastrin 17 antibodiesin a human patient, the hormone gastrin 17 and the prohormone progastrinG17-Gly are neutralized in vivo, so as to inhibit their physiologicaleffects. In particular, the neutralization of G17 and the precursorG17-Gly prevents the binding of these peptides to their physiologicalreceptors, thereby inhibiting the growth of the tumor cells.

The anti-G17 immunogens, comprise fragments of the N-terminal aminoacids of G17 conjugated to an immunogenic carrier such as Diphtheriatoxoid (DT), by a spacer peptide, and raise antibodies which bind boththe amidated and glycine-extended forms of G17.

In one embodiment of the invention, the method of immunization againstamidated or glycine-extended G17 comprises active immunization, whereina patient is immunized with an immunogen of the invention. The immunogenstimulates the production of antibodies against amidated andglycine-extended G17 in the immunized patient, inducing sufficientantibody titers to neutralize and inhibit the physiological effects ofamidated and glycine-extended G17 so as to limit the cancer-trophichormone levels produced by the patient. The physiological neutralizationof progastrin G17-Gly hormone by the anti-G17 antibodies produced in thepatient inhibits the growth of tumor cells dependent on progastrinG17-Gly as the growth stimulator or inducer. The treatment methods ofthe invention are particularly suited for the treatment of G17-Gly oramidated G17-responsive gastrointestinal cancers.

The immunogens of the invention comprise peptides composed of twofunctional regions: an immunomimic region and a spacer region. Thefunction of the immunomimic region which immunologically crossreactswith G17 and G17-Gly, is to induce antibodies in the immunized animalthat bind to the targeted G17 hormone, i.e. amidated and glycineextended G17, thereby inhibiting G17 function and arresting or slowingthe growth of the G17-dependent tumor cell. The present immunogensinduce a biologically effective immune response following administrationof the immunogen in all immunized animals tested. The immunomimicpeptide-spacer of this invention can be coupled to immunologicalcarriers over a wide range of peptide to carrier substitution ratios andyield effective immunogens.

In another embodiment of the invention, the method of treatmentcomprises passive immunization, in which antibodies against G17 areadministered to the patient in a sufficient concentration to reduce thelevels of circulating unbound G17 and G17-Gly. The reduced levels offree G17 and progastrin in the circulating blood of a patient as aresult of anti-G17 antibody administration, results in an inhibition ofthe growth of the tumor cells, thereby stopping or reducing the growthand size of the tumors. In a preferred embodiment of this aspect of theinvention, the anti-G17 antibodies for human therapy may be chimeric,humanized, or human monoclonal antibodies which may be produced bymethods well known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatical representation of glycine-extended G17,carboxy-amidated G17 and an anti-G17 immunogen containing an aminoterminal portion of G17.

FIG. 2 is a graphic representation of the displacement of [¹²⁵I]G17 fromrabbit anti-human G17(1-9): DT (N-terminal specific) antiserum by G17,glycine-extended G17 and G34.

FIG. 3 is a graphic representation of the displacement of [¹²⁵I]G17 fromrabbit anti-human G17 (C-terminal specific) antiserum by G17,glycine-extended G17 and G34.

FIG. 4 depicts a bar graph on the effect of immunizations with theimmunogens of the invention on the median cross-sectional areas ofDHDK12 tumors (inter-quartile ranges for each median are present at thetop of the respective columns).

FIG. 5 depicts a bar graph on the effect of immunizations with theimmunogens of the invention on the final median weights of DHDK12 tumors(interquartile ranges for each median are present at the top of therespective columns).

FIG. 6 depicts the anti-rat G17 antibody levels of individual G17(1-9)-and DT-immunized rats (measured at a 1:100 dilution of sera). 1 At tumorchallenge (rat G17(1-9) treatment, 5 immunizations) 2 At therapytermination (rat G17(1-9) treatment, 7 immunizations) 3 At tumorchallenge (DT treatment, 5 immunizations) 4 At therapy termination (DTtreatment, 7 immunizations) 5 Positive control (rat anti-rat G17:DTantiserum) 6 Negative control (normal rat serum)

Antibody levels were measured by an ELISA capture assay in whichanti-rat G17:DT antibodies bound to rat G17-BSA coated on 96 wellmicrotiter plates. Antibody binding was detected using an alkalinephosphatase based method with pNPP as substrate.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the invention are directed to administering to a patientan anti-G17 immunogen which induces antibodies in the immunized patientwhich bind and neutralize amidated-G17 and glycine-extended G17 (seeFIG. 1).

Surprisingly, the immunogens and immunogenic compositions against G17disclosed in co-assigned U.S. Pat. Nos. 5,023,077 and 5,468,494, alsoproduce antibodies in immunized animals which react with and neutralizeamidated gastrin 17 and glycine-extended gastrin 17. Advantageously,therefore, these immunogens may be used in methods of treating cancerdisease states which are trophic due to these precursor hormones.

U.S. Pat. Nos. 5,023,077 and 5,468,494, the disclosures of which arehereby incorporated by reference in their entirety, disclosecompositions containing anti-gastrin 17 immunogens and methods of usingthese compositions for the treatment of gastric and duodenal ulcers andgastrin induced cancers. The present invention concerns the use of thesame anti-G17 immunogens to treat disease states such asgastrointestinal cancers which are affected by the prohormone G17-Gly.

In the present invention, a serum sample from the patient having agastrointestinal cancer can be assayed to determine the level of G17-Glyin the patient's blood. An effective dosage ranging from 0.001 to 2 mgof the immunogenic composition is administered to the patient for thetreatment of the gastrointestinal cancer. The effective dosage of theimmunogenic composition should be capable of eliciting an immuneresponse in a patient of effective levels of antibody titer against bothhuman gastrin 17 and the G17-Gly within 1-3 months after immunization.Following the immunization of a patient, the antibody titer levelsagainst amidated or glycine-extended G17 are monitored from a sample ofblood taken from the patient, and booster immunizations should be givenas required to maintain an effective antibody titer which willneutralize G17-Gly and amidated G17. The effective antibody titer whichwill neutralize G17-Gly and amidated G17 is defined as the minimumantigen binding capacity of 5 picomoles of antigen-bound in onemilliliter of the patient's serum, as measured by standard immunologicalassays. In addition, serum G17-Gly can be monitored to assess theeffectiveness of immunization against G17. Effective treatment ofgastrointestinal cancers such as colorectal adenocarcinomas, accordingto this method should result in inhibition of tumor growth and adecrease in size of the tumor.

The antibody titers raised by the anti-G07 immunogens are in excess ofthose required to neutralize serum G17 resulting in high serum levels ofuncomplexed antibodies which are free to bind to G17-Gly. Thus, the‘free’ serum-associated antibodies would be available to neutralizecell-associated G17 peptides in well-vascularized areas of the tumors.

Antibodies raised by the anti-G17 immunogens of the present inventionmay have significant anti-trophic effects against gastrointestinalcancer, such as a colon tumor by two potential mechanisms: (i)neutralization of serum G17, and (ii) neutralization of cell-associatedprecursor gastrin molecules.

The following examples demonstrate the effect of active immunizationwith rat G17 immunogen on the in vivo growth of the rat colon cancerline, DHDK12. DHDK12 is a rat colonic tumor cell line of epithelialmorphology (Martin et al. 1983). The immunogen tested is composed of theN-terminal 9 amino acids of G17 linked to DT by a spacer peptide, andcan be made specific for either human or rat G17. Antiserum raised byanti-G17 immunization is denoted as anti-G17(1-9):DT and contains aspacer peptide.

EXAMPLE 1

These experiments demonstrate that the immunogen induces antisera thatbind to amidated G17 and glycine-extended G17, but not to G34.

Gastrin-Specificity of Antiserum Raised by Anti-G17 Immunization ofRabbits

Antisera were absorbed onto a solid phase at a concentration of 100μg/ml and displacement was determined in a competitive assay with afixed concentration of radiolabelled G17 (1000 pg/ml) and increasingconcentrations of unlabelled ligands (1-25,000 pg/ml).

FIGS. 2 and 3 show the displacement of [¹²⁵I]G17 from rabbit anti-humanG17 antiserum by G17, G17-Gly and G34. The antiserum used in the testdepicted in FIG. 2 was obtained from animals immunized with G17(1-9):DTand was specific for the N-terminal end of G17; the antiserum for FIG. 3was specific for the C-terminal end of G17. G17 displaced radiolabelledG17 from both antisera preparations with a 50% inhibitory concentration(IC₅₀) of 3500 pg/ml for the rabbit anti-human G17 (1-9):DT (N-terminal)and 800 pg/ml for the rabbit anti-G17 (C-terminal). Glycine-extended G17did not displace radiolabelled G17 from the C-terminal specificantiserum, but did from the N-terminal specific antiserum (IC₂₅12,000pg/ml), demonstrating that the glycine-extended G17 binds to N-terminalspecific antiserum, but not to C-terminal specific antiserum. G34displaced radiolabelled G17 from the C-terminal (IC₂₅ 500 pg/ml), butnot the N-terminal specific antiserum, demonstrating the specificity ofthe G17(1-9):DT antiserum for G17 and glycine-extended G17 and not toG34.

EXAMPLE 2

These experiments show that the DHDK12 rat colonic cells produceglycine-extended gastrin 17 and that anti-G17 antiserum reduces thelevels of precursor gastrin produced by the cells.

Radioimmune Assay of Precursor Gastrin Levels DHDK12 cells were grown tosub-confluence in RPMI 1640 culture medium (Gibco, Irvine, Scotland, UK)supplemented with 2 mM glutamine (Sigma, Poole, Dorset, UK) and 10%heat-inactivated foetal calf serum (FCS, Sigma). The cells wereincubated in humidified conditions at 37° C. with 5% CO₂. Cells wereharvested with 0.025% EDTA (15 minutes at 37° C.), washed bycentrifugation and 2×10⁶ cells seeded into flasks containing serum-freemedium (RPMI 1640 in a 1:1 ratio with Hams F12 (Gibco) with 0.5% bovineserum albumen [BSA]). Cells were harvested with 0.025% EDTA, washed,re-suspended in 1 ml of sterile distilled water and heated in a boilingwater bath. The levels of glycine-extended gastrin were measured byradioimmunoassay (RIA) using antibodies 109-21 and L-2 as described(Nemeth et al. 1993).

Levels of Gastrin Precursors Associated with DHDK12 Cells

DHDK12 cells were shown to contain glycine-extended gastrin, but notamidated G17, in two separate experiments as shown in Table 1. TABLE 1Precursor gastrin levels associated with DHDK12 cells Glycine-extendedG17 conc. Amidated G17 conc (fmol/10⁷ cells) (fmol/10⁷ cells) Experiment1 31.2 ND¹ (1.0 × 10⁷ cells/ml) Experiment 2 80.0 ND¹ (1.27 × 10⁷cells/ml)Tumor cell extracts were prepared by heating cells in 1 ml of sterilewater. Cell extracts were recovered by centrifugation and progastrin,glycine-extended gastrin and amidated G17 levels were measured usingantibodies 109-21 and L-2 respectively, as previously described (Nemethet al. 1993).¹ND - Not detectedEffect of Rabbit Anti-ratG17:DT Treatment on the Precursor GastrinLevels of DHDK12 cells

Semi-confluent DHDK12 cell monolayers were prepared as describedpreviously in serum-free medium and harvested with 0.025% EDTA. Affinitypurified rabbit anti-ratG17:DT and rabbit anti-DT (negative control)were then added to the flasks at equivalent protein concentrations togive an antigen binding capacity for the former of 3 ng/ml. The cellswere incubated for 4 days after which cell extracts were prepared andassessed for precursor gastrin levels by the RIA described above.

The effect of in vitro treatment with affinity purified rabbitanti-ratG17 (1-9):DT and rabbit anti-DT antisera on the precursorgastrin levels associated with DHDK12 cells in serum-free medium isshown in Table 2. TABLE 2 Precursor gastrin levels of DHDK12 cells afterin vitro treatment with rabbit anti-G17(1-9):DT antiserumGlycine-extended G17 conc. Amidated G17 conc Treatment (fmol/10⁷ cells)(fmol/10⁷ cells) Rabbit ND¹ ND¹ anti-G17(1-9):DT antiserum Rabbitanti-DT 67.0 ND¹ antiserumDHDK12 cells were grown in serum-free medium (RPMI 1640 in a 1:1 ratiowith Hams F12 with 0.5% bovine serum albumen). Affinity purified rabbitanti-rat G17(1-9):DT and rabbit anti-DT were then added to the flask ata protein concentration of 3 ng/ml and incubated for 4 days. Cellextracts were recovered by centrifugation and progastrin,glycine-extended gastrin and amidated G17 levels were measured usingantibodies# 109-21 and L-2 respectively, as previously described (Nemeth et al.1993).¹ND Not detected

As can be seen in Table 2, rabbit anti-ratG17:DT antiserum reduced thelevels of glycine-extended G17 from 67 pg/ml to undetectable.

DHDK12 cells were also shown to express cell-associated glycine-extendedG17 but not amidated gastrin. In vitro treatment of DHDK12 cells withrabbit anti-ratG17 (1-9):DT reduced the levels of cell-associatedprecursor gastrin when compared to cells treated with rabbit anti-DTcontrol antiserum. Thus, antibodies produced by anti-G17 immunizationmay interrupt an autocrine growth loop involving such peptides as aconsequence of down-regulation of gastrin translation.

EXAMPLE 3

The following experiments demonstrate that immunization of rats with theRat G-17 (1-9) DT immunogen markedly inhibits the growth of DHDK12tumors in vivo.

Experimental Animals

Male BDIX rats (The Animal Unit, University of Liverpool, UK) of age6-10 weeks weighing 340-430g were housed in pairs and maintained in acycle of 12 hours light and 12 hours dark at 25° C. with 50% humidity.The rats were allowed to acclimatize for at least 7 days before use.

Immunization Procedure

Rat G17(1-9) coupled to DT or the DT component alone were dissolved insterile saline (0.9%), pH 7.3 at 1 mg/ml. The adjuvant nor-muramyldipeptide (nor-MDP, Peninsula Labs., CA) was added to the conjugate togive a final concentration of 5001 g/ml. The aqueous solution was mixedwith oil (Montanide ISA 703 AMS Seppic, Inc., Paris, France) in a 1:1ratio (vol:vol) and placed in a glass syringe which was attached to asecond syringe with a three-way stopcock as connector and the mixtureforced back and forth through the syringes 100 times (the stopcockproduced a right angle shear to assist emulsification).

Control animals received an identical emulsion containing the DT peptideonly, and all experimental groups were equalized with respect to weight.A 200 μl volume of the emulsion was injected subcutaneously (s.c.) inthe right hand flank of the experimental animal. The animals wereimmunized at 21 day intervals and the tumor implanted after 5immunizations.

Initiation of Tumor Growth

DHDK12 cells were suspended in sterile 0.9% saline at a concentration of2.5×10⁷/ml. Rats were anaesthetized by a 1 ml injection of Hypnorm(Fentanyl citrate 0.315 ng/ml and Fluanisone 10 mg/ml, Jannsen,Belgium), Hypnovel (Midazolan 5 ng/ml, Roche, Switzerland) and steriledistilled water in a 1:1:5 ratio. Following a s.c. incision on the rightflank, a 200 $1 volume of the cell suspension was injected into themuscle layer of the abdominal wall and the surgical incision closed witha wound clip. Each experimental group consisted of between 16-18 rats.

Effect of Rat Anti-G₁₇ Immunization on the in Vivo Growth of DHDK12Tumors

FIGS. 4 and 5 show the effect of immunization with rat G17(1-9)-DTimmunogen (5 immunizations prior to injection of cells) on the finalcross-sectional areas and weights, respectively, of DHDK12 tumors. Thetumors had significantly reduced cross-sectional areas in rats immunizedwith anti-G17 immunogen. FIG. 4 illustrates data which show that themedian cross-sectional areas of tumors from anti-G17 treated rats werereduced by 70.2% when compared to tumors from the DT controls, p=0.005,Mann Whitney. DHDK12 tumors also had significantly reduced tumor weightsin rats immunized with anti-G17 immunogen. FIG. 5 shows that DHDK12tumor weights were reduced by 56.5% when compared to tumors from the DTcontrols, p=0.0078. The mean animal weight in the anti-G17 treated ratsrose from 399 g to 452 g (13% increase) over the duration of theexperiment and in the DT-treated animals from 392 g to 447 g (13.8%increase) indicating that the growth rate of the animals was notaffected by administration of the G17(1-9)-DT immunogen.

EXAMPLE 4

The experiments show the levels of anti-rat G17 antibodies induced inimmunized rats that were implanted with DHDK12 tumors.

Anti-rat G17 Antibody Levels of Rat G17(1-9): DT-immunized Rats

To determine the antibody response to the emulsified rat G-17(1-9)DTimmunogen, rats were tail-bled at various time points and an ELISAtechnique was used to determine the anti-rat G17:DT antibody titers.

A rat G17-BSA conjugate was prepared at a concentration of 2 μg/ml inGlycine buffer (0.1 M, pH 9.5) and 25 μl was plated per well into96-well Immunlon U plates (Dynatech Labs., Sussex, UK) and incubatedovernight at 4° C. The unabsorbed conjugate was then flicked out and thewells washed in buffer which consisted of 0.9% saline, pH 7.3 containing0.5% Tween-20 (Sigma) and 0.02% NaN₃ (Sigma). This buffer was used forboth washing and reagent dilutions. The test sera (from animalsimmunized with the rat gastrin immunogen) were used at a startingdilution of 1:100 and at 10 fold dilutions thereafter. The positivecontrol was rat anti-rat G17 antiserum from previously immunized animalsand the negative controls were normal rat serum and sera from ratsimmunized with DT. These were used at the same dilutions as describedfor the test sera. The test and control sera were added to the wells in25 μl volumes either in the absence or presence of 25 μl/well ratG17-BSA at 100 μg/ml (control wells received 25 μl assay buffer). Theplates were then incubated for 60 minutes at room temperature. Theplates were washed with saline buffer, then goat anti-rat Ig(H+L)-biotin (Zymed, San Francisco, Calif.) was added to the wells at a1:500 dilution, 50 μl/well, and incubated for 60 minutes in the dark atroom temperature. The plates were washed with saline buffer and avidinalkaline phosphatase (Zymed) was added to wells at a 1:100 dilution, 50μl/well and incubated for 60 minutes in the dark at room temperature.After washing with saline buffer, p-nitro-phenylphosphate (PNPP)substrate (Sigma) in substrate buffer was added to the wells at 50μl/well and after 5 minutes developing time the absorbance was read at405 nm. The difference in absorbance between untreated sera and seraco-incubated with rat G17-BSA was calculated as the specific absorbance.

The free anti-rat G17(1-9):DT antibody levels (those in excess of theantibodies required to bind serum-associated G17) were measured and areexpressed as the specific absorbance obtained at a 1:100 dilution ofserum (FIG. 6). After 5 immunizations, at the time of tumor cellinjection, the mean antibody level was 0.243 absorbance units (Group 1in FIG. 6). The mean antibody level had increased, by the termination ofthe study, following 2 further immunizations, to 0.66 absorbance units(Group 2 in FIG. 6) and were in the range of the positive control (Group3 in FIG. 6). Antibody levels from animals immunized with DT had a meanabsorbance of 0.1 units (Groups 4 and 5 in FIG. 6) and the negativecontrol (normal rat serum) showed no absorbance (Group 6 in FIG. 6).There was no apparent correlation between tumor weight and antibodylevels as measured by a Linear Regression Analysis, (p=0.14).

EXAMPLE 5

The following experiments show that immunization against G17 reducedserum G17 levels and that these reduced levels correlated with reducedtumor growth.

G17 Levels in the Immunized Rats as Determined by an Inhibition RIA

Rabbit anti-G17 antiserum (C-terminal-specific, Dakopatts, Bucks., UK)was coated onto 96-well microtitre plates at a protein concentration of10 ng/well in PBS. A standard curve was constructed by incubating[¹²⁵I]G17 at a fixed concentration of 10,000 CPM/well with increasingconcentrations of G17.

The unknown samples containing free gastrin, bound G17, free and boundanti-G17 antibodies were prepared in 250 μlaliquots. A 125 μl aliquot ofnewborn calf serum (Sigma) and 312.5 μl of 25% polyethylene glycol(Sigma) were added to each sera sample. These were vortexed and spun at1500 rpm for 30 minutes. The supernatant was removed and boiled (toensure no free antibodies remained) and was classified as the freegastrin sample.

The pellet was washed 5 times in 0.002 M veronal buffer (pH 8.4)containing 0.5% bovine serum albumen and solubilized by boiling in 250μl of water. This was classified as containing bound gastrin. Triplicatealiquots of each of the samples were added to the labelled G17 and thelevel of inhibition was determined. Gastrin levels in the rat sera werethen calculated from the standard curve.

The free serum gastrin level (as measured with an antiserum directedagainst the carboxy terminus of G17) of the DT-immunized rats was foundto be 114.0 pg/ml (standard deviation of 31) compared to 68.5 pg/ml(standard deviation of 20) in the rat G17(1-9): DT-immunized group. Thiscorresponds to a 40% reduction in total gastrin. Total serum gastrinlevels were correlated with final tumor weight and the correlationcoefficient was found to be statistically significant, (p=0.011, LinearRegression Analysis. The levels of serum gastrin bound to antibodies wasfound to be zero in the DT-immunized rats and ranged from 30.1 to 253.7pg/ml in the rat G17(1-9): DT-immunized rats (median of 53.3 pg/ml).

DHDK12 rat colon tumor cells growing in vivo, by virtue of theirCCKB/gastrin receptors, have responded to serum G17. In theseexperiments, excess anti-G17 antibodies (i.e. those not bound to serumG17) were measured during the tumor challenge. The total serum gastrinlevels were shown to be reduced by 40% and a significant positivecorrelation was shown between tumor weight and serum gastrin levels atthe termination of the therapy. In addition, antibody-bound gastrin wasalso detected in the rat G17(1-9): DT immunized, but not in theDT-immunized, rats. Thus, neutralization of serum-associated gastrincontributed to reduced tumor growth.

EXAMPLE 6

The experiments show that imminization against G17 affects thehistological appearance of DHDK12 tumors.

Histological Evaluation of the Rat Tumors

At termination of therapy, the DHDK12 tumors were fixed in 10% formalcalcium and embedded in paraffin. 5 μm sections were cut on a cryostat,stained with Haematoxylin and Eosin and the pathological parameters ofthe tumors assessed independently by a Pathologist. Image analysis wasperformed on the tumor sections using a Seescan Image Analyzer in ablind manner to assess the area of viable tumor tissue.

Gastrin receptors (GR) were detected using a rabbit anti-CCKB/gastrinreceptor polyclonal antiserum. Sections were incubated with a 1:500dilution overnight at 4° C. Binding was detected using the avidin-biotintechnique with immunoperoxidase as the enzyme tracer anddiaminobenzidene as the substrate.

Histological evaluation revealed that the tumors from rat G17(1-9):DT-immunized rats had a smaller rim of viable tumor tissue around theleading edge of the tumor and a greater degree of central necrosis whencompared to tumors from rats immunized with DT. This was quantified byimage analysis and the mean percentage of viable cell area in tumorsfrom rat G17(1-9):DT-treated rats was 40.3% (standard deviation of 9.1)compared to 58.6% (standard deviation of 10.4) for the DT-immunized rats(p=0.003, Student's t test).

Higher magnification microscopy showed that the tumor cells in theDT-immunized rats grew in a regular trabecular manner, whereas the tumorcells from G17(1-9):DT-immunized rats had a disrupted pattern of growth.There was also more connective tissue in the tumors from G17(1-9):DT-treated rats when compared to tumors from DT-treated rats (connectivetissue:tumor ratio being 75:25 and 50:50, respectively). Areas of focalnecrosis were present within the viable tumor tissue in the G17(1-9):DTtreated group and also an increased inflammatory infiltrate whichappeared to be composed mostly of lymphocytes. The tumors from rats inboth treatment groups were stained with anti-GR antiserum and it wasshown that the viable cells remaining in both the DT and the G17(1-9):DTtreated groups had retained their GR positivity.

Immunization with the G17:DT immunogen reduces the in vivo growth ofDHDK12 rat colon tumors as shown by both cross-sectional area and weightmeasurements. Extrapolation of quantitative assessment of viable tumortissue by image analysis indicated that the weight of viable tumortissue may have been reduced by as much as 68%.

One further finding was the focal areas of necrosis within the tumortissue in anti-G17 treated rats and the presence of an inflammatoryinfiltrate in certain areas of the tumor which was mainly composed oflymphocytes. One possible explanation of such findings is that anantibody-dependent cellular cytotoxic response was instigated by theanti-G17 immunogen. The mechanism of such a response as applied toimmunization against G17 is unknown.

Anti-G17 immunization resulted in the potential neutralization of twotrophic forms of gastrin, G17 and glycine-extended G17, and thus mayinduce cytostasis within the tumors. The histological observationsprovide evidence for the theory that tumors from anti-G17 immunogentreated rats have a slower growth rate than tumors from control rats asthe growth pattern, the degree of fibrosis and the area of the viabletumor tissue was significantly reduced in the former rats.Interestingly, the viable tumor cells remaining were shown to maintaintheir expression of GR. This indicates that in this tumor model, thegastrin hormone-sensitive phenotype may have been expressed by all thecell clones and there was no outgrowth of gastrin hormone-insensitiveclones leading to escape from anti-G17 immunogenic inhibition.

EXAMPLE 7

Immunocytochemical Evaluation of the CCKB/Gastrin Receptor Expression ofDHDK12 Cells

DHDK12 cells were suspended at a concentration of 1×10⁶ /ml and 200 μlvolumes were cytospun onto microscope slides (1200 rpm, 5 minutes). Thecells were fixed with methanol at −20° C. (5 minutes) and permiabilizedby treatment with graded alcohols. The cells were incubated with therabbit anti-CCKB/gastrin receptor antiserum and stained as previouslydescribed.

CCKB/gastrin receptor expression of DHDK12 cells was evaluated withantiserum raised against peptide sequences derived from the humanCCKB/gastrin receptor. DHDKi2 cells showed a strong and specificmembrane-associated immunoreactivity indicative of a high level ofgastrin receptor expression. Cells treated with a control rabbitantiserum showed no specific immunoreactivity.

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1. A method for the treatment of glycine-extended gastrin-17-dependentgastrointestinal tumors, comprising administering to a mammal atherapeutically effective amount of an anti-G17 immunogenic composition.2. The method of claim 1, wherein the immunogen induces anti-G17antibodies of an effective titer in the immunized mammal which bind andneutralize amidated and glycine-extended gastrin-17.
 3. The method ofclaim 1, wherein the gastrointestinal tumors containgastrin/cholecystochinin B receptors.
 4. The method of claim 1, whereinthe gastrointestinal tumors are colorectal adenocarcinomas.
 5. Themethod of claim 1, wherein the mammal is a human.